Immobilization Of Α-amylase Research Articles

Improved performance of α-amylase immobilized on poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads

α-Amylase was successfully immobilized onto poly(glycidyl methacrylate-co-ethylenedimethacrylate) (PGMA/EDMA) beads with high immobilization efficiency of 87.8%. PGMA/EDMA beads with a relatively uniform diameter of 2–3μm were prepared by single-step swelling polymerization. After amination with ethanediamine and activation with glutaraldehyde, PGMA/EDMA beads showed commendable α-amylase immobilization capacity of 35.1mgg−1 carrier. Compared with free form, immobilized α-amylase had increasement of 12.94mgmL−1 for Km and 0.124mmolmL−1min−1 for Vmax, improved acid resistance (the optimal pH from 7 to 5), presented better thermal stability by retaining 61% activity than 40% at 90°C, and displayed high operational reusability by retaining 58% of its initial activity after nine uses. Moreover, less than 10% of the free enzyme and more than 80% of the immobilized enzyme retained activity after 180min pre-incubation at 50°C. The easy modification, high immobilization efficiency and good properties of immobilized α-amylase in the present study indicate that PGMA/EDMA beads are suitable for α-amylase immobilization. The enhancement of acid resistance and thermo stability is doubtless of benefit for the industrial use of α-amylase.

INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND α-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES

Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens α-amylase was immobilized on SnO2 nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90% of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of α-amylase. The genotoxicity of SnO2-NPs was analyzed by pUC19 plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO2-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed α-amylase was 5°C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses.

Use of Agro Waste Biomass for α-Amylase Production by Anoxybacillus amylolyticus: Purification and Properties

Knowledge accumulated from fundamental and applied studies on Anoxybacillus suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, waste treatment, enzyme technology, and bioenergy production. We investigated the purification, biochemical characterization and immobilization of a thermostable α-amylase from the thermophilic Anoxybacillus amylolyticus, strain MR3CT, isolated in Antarctica and its production on vegetable wastes. In particular, the rhizome from Arundo donax, waste biomass of Cynara cardunculus and potato peels were tested either in Submerged Fermentation (SmF) and Solid State Fermentation (SSF) conditions. The amylase from A. amylolyticus, with a molecular weight of about 60 kDa, displayed an optimum enzyme activity at 60°C and pH 5.6. Moreover, by retaining up to 70% of total activity after 48 h at 60°C, it showed high thermostability in the presence of 2 mM calcium ion. The immobilized enzyme maintained the 48% of its initial activity after the sixth reuse. The optimal conditions for its production in SmF were achieved at 60°C for 24 h with 1% of rhizome from Arundo donax, which was about 2126 U/gds. SSF cultures reached maximum α-amylase yield (102 U/gds) when grown on waste biomass of Cynara cardunculus as substrate, with a substrate-water ratio of 1:1 (w/v), and incubation at 60°C for 4 days. In this study, rhizome of A. donax resulted to be a good substrate for amylase production in SmF thus allowing a cheaper alternative to obtain amylolytic enzymes. Indeed by using rhizomes from A. donax as growth substrate it was possible to recovery an amylase activity level higher than that obtained by synthetic complex medium. Amylase production was also investigated under SSF conditions by using the above listed wastes as sole carbon source for A. amylolyticus growth. Under these conditions, C. cardunculus gave a higher enzyme yield per reactor volume.

Immobilization of α-amylase purified fromBacillus cereusMTCC 10205 by entrapment and adsorption on various support systems

Alpha-amylase from Bacillus cereus MTCC 10205 was immobilized by entrapment in alginate beads and carrageenan as well as via adsorption on charcoal. The immobilized enzyme had the pH optima 6.0 (in comparison to 5.5 of free enzyme), and temperature optima of 60°C (5°C higher than that of soluble enzyme). Thermostability of enzyme increased remarkably after immobilization. Storage stability and Km of the immobilized enzyme was higher than that of free form. Alginate entrapped enzyme could be stored at 4°C for one week without appreciable loss in activity, however carrageenan entrapped and charcoal adsorbed enzyme was stable only upto four days. The alginate entrapped α-amylase was found to have good operational utility (reused upto seven cycles) while carrageenan entrapped and charcoal adsorbed enzyme were reusable upto three cycles without appreciable loss in activity. Results of the study indicated that alginate entrapped enzyme had better thermostability and operational utility.

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles

In this work, α-amylase enzyme was covalently immobilized on the surface of silica-coated modified magnetite nanoparticles, for the first time. The synthesis and immobilization process is simple and very fast and consists of the following steps: (1) preparing the magnetic iron oxide nanoparticles using the co-precipitation method, (2) coating NP with silica (SiO2) by sol–gel reaction, (3) preparing the amino-functionalized magnetite NPs by treating silica-coated NPs with 3-aminopropyltriethoxysilane, (4) activating immobilization of α-amylase on the activated amino-functionalized magnetite NPs, and (5) covalently immobilization of α-amylase on the activated-amino-functionalized magnetite NPs. The synthesis steps and characterizations of NPs were examined by FT-IR, XRD, EDX and TEM. The optimum concentration and time for maximum enzyme activity of the immobilized α-amylase are identified to be 150μg and 4h, respectively for the hydrolysis of starch. The immobilized α-amylase showed maximal catalytic activity at pH=6.5 and 45°C. The kinetic studies shows overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Similarly, the thermal stability of the enzyme is found to increase after the immobilization. The Immobilized α-amylase has also been demonstrated to be capable of being reused for six cycles while retaining ∼85% of the initial activity. By using a magnetically active support, quick separation of amylase from reaction mixture is enabled. The Km values were found as 6.27 and 4.77mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 2.44 and 11.58μmol/mgmin, respectively.

Immobilization of α-amylase on gum acacia stabilized magnetite nanoparticles, an easily recoverable and reusable support

Abstract In this work, α-amylase is immobilized, using glutaraldehyde, onto magnetite nanoparticles prepared using gum acacia as the steric stabilizer (GA-MN), for the first time. The immobilization of amylase to GA-MN is very fast and the synthesis of GA-MN is very simple. The use of GA enables higher immobilization of α-amylase (60%), in contrast to the unmodified magnetite nanoparticles (∼20%). The optimum pH and temperature for maximum enzyme activity for the immobilized amylase are identified to be 7.0 and 40 °C, respectively, for the hydrolysis of starch. The kinetic studies confirm the Michaelis–Menten behavior and suggests overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Similarly the thermal stability of the enzyme is found to increase after the immobilization. The GA-MN bound amylase has also been demonstrated to be capable of being reused for at least six cycles while retaining ∼70% of the initial activity. By using a magnetically active support, quick separation of amylase from reaction mixture is enabled. The catalytic rate of amylase is actually found to enhance by twofold after the immobilization, which is extremely advantageous in industry. At higher temperature, the immobilized enzyme exhibits higher enzyme activity than that of the free enzyme.

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration

α-Amylase was immobilized on Dowex MAC-3 with 88 % yield and amyloglucosidase on Amberlite IRA-400 ion-exchange resin beads with 54 % yield by adsorption process. Immobilized enzymes were characterized to measure the kinetic parameters and optimal operational parameters. Optimum substrate concentration and temperature were higher for immobilized enzymes. The thermal stability of the enzymes enhanced after the immobilization. Immobilized enzymes were used in the hydrolysis of the natural starch at high concentration (35 % w/v). The time required for liquefaction of starch to 10 dextrose equivalent (DE) and saccharification of liquefied starch to 96 DE increased. Immobilized enzymes showed the potential for use in starch hydrolysis as done in industry.

A novel reusable PAni-PVA-Amylase film: Activity and analysis

Alpha-amylase was immobilized onto polyaniline-polyvinyl alcohol (PAni-PVA) film by cross linking with glutaraldehyde. The activity yield and immobilization efficiency was calculated as 13.58% and 30.60% respectively. The PAni-PVA-Amylase films were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and amylase activity assay. The immobilized α-amylase had its optimum activity at pH 7.0 and 25°C. The immobilization of α-amylase onto the PAni-PVA films protected the enzyme from Fe2+ inhibition. The PAni-PVA-Amylase films could be used effectively for 30 cycles with 60% retention of the activity.

Preparation of a magnetically recoverable biocatalyst support on monodisperse Fe3O4 nanoparticles

A magnetically recoverable biocatalyst has been effectively prepared through the immobilization of α-amylase onto the Fe3O4 nanoparticles. The magnetic nanoparticles (MNPs) were synthesized by a sol–gel method in an aqueous system. The MNPs’ surfaces were modified with sodium silicate and 3-aminopropyltriethoxysilane (APTS). The adsorptive immobilization of α-amylase was attempted onto the APTS–SiO2–Fe3O4 nanoparticles. The physicochemical properties of the biocatalysts were characterized with Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD) and thermal gravimetric analysis (TGA). The magnetic properties of the biocatalyst were analyzed by a vibrating sample magnetometer (VSM). The activity of free and immobilized α-amylase was also evaluated by direct measurement of the absorbance of starch solution at 620 nm using UV-vis spectrophotometry. The prepared particles exhibited superparamagnetic behavior at room temperature. The mass saturation magnetization (Ms) of the bare Fe3O4 nanoparticles and the biocatalyst was 175.7 and 50.4 emu g−1 respectively. A biocatalyst was produced with a loading capacity of α-amylase of about 235 mg g−1. The enzymatic activity of the immobilized enzyme was 882 U g−1 which is about 79.53% of the free enzyme activity when not involved in the immobilized system. The immobilized enzyme exhibited significant thermal stability (stable up to 70 °C) and good durability (recycled 3 times without any obvious loss of enzymatic activity). This synthetic method provides a possible industrial route for the use of enzyme catalysts which eliminate the high operational costs and complicated recycle/reuse problems in the conventional enzyme catalysis process.

Polyaniline-assisted silver nanoparticles: a novel support for the immobilization of α-amylase

The size distribution of nanomaterials and their similarity in size with enzyme molecules together with other advantageous properties such as thermal stability, high surface-to-volume ratio, and irradiation resistance have revolutionized nanobiocatalytic approaches in various areas of enzyme technology. In the present study, polyaniline-assisted Ag nanocomposites were synthesized using ammonium peroxydisulfate as oxidant. These nanocomposites were used as a support for the covalent conjugation of α-amylase, one of the important industrial enzymes. X-ray diffraction study showed that the crystalline nature of nanocomposites was increased in the presence of Ag nanoparticles. Thermogravimetric and differential thermal analysis revealed that the synthesized nanocomposites retained significantly very high thermal stability. Scanning electron micrograph showed that Ag nanoparticles were homogeneously dispersed in polyaniline film providing large surface area and microenvironment for enzyme loading. Fourier transform infrared spectroscopy confirmed the conjugation of α-amylase to the functionalized nanocomposites. The conjugated α-amylase exhibited better tolerance to variations in the medium pH and temperature compared with the native enzyme. Immobilized α-amylase hydrolyzes starch more efficiently as compared to the free enzyme in batch process.

α-Amylase immobilization on gelatin: Optimization of process variables

α-Amylase was extracted and purified from soybean seeds to apparent homogeneity by affinity precipitation. The homogeneous enzyme preparation was immobilized on gelatin matrix using glutaraldehyde as an organic hardener. Response surface methodology (RSM) and 3-level-3-factor Box–Behnken design was employed to evaluate the effects of immobilization parameters, such as gelatin concentration, glutaraldehyde concentration and hardening time on the activity of immobilized α-amylase. The results showed that 20% gelatin (w/v), 10% glutaraldehyde (v/v) and 1 h hardening time yielded an optimum immobilization of 82.5%.

Immobilization of porcine pancreatic α-amylase on magnetic Fe2O3 nanoparticles: Applications to the hydrolysis of starch

Enzymes play a pivotal role in catalyzing diverse reactions. However, their instability upon repetitive/prolonged use, as well as their inhibition by high substrates and product concentration, remains an area of concern. In this study, porcine pancreatic α-amylase was immobilized on magnetic Fe2O3 nanoparticles (Fe2O3-NPs) in order to hydrolyze starch. The magnetic nanoparticle bound enzymes retained 94% of their initial enzyme activity. X-ray diffraction and atomic force microscopy analyses showed that the prepared matrix had advantageous microenvironment and a large surface area for binding significant amounts of protein. Functional groups present in enzyme and support were monitored by Fourier transform infrared spectroscopy. Immobilized enzyme exhibited lowered pH optimum (pH 6.0) to a greater degree than its soluble counterpart (pH 7.0). Optimum temperature for the immobilized enzyme shifted towards higher temperatures. The immobilized enzyme was significantly more resistant to inactivation caused by various metal ions and chemical denaturants. Immobilized α-amylase hydrolyzed 92% starch in a batch process, after 8 h at 40°C; while the free enzyme could hydrolyze only 73% starch under similar experimental conditions. A reusability experiment demonstrated that the immobilized enzyme retained 83% of its original activity even after its 8th repeated use.

Preparation of Magnetic Nanoparticles and Their Use for Immobilization of C-Terminally Lysine-Tagged Bacillus sp. TS-23 α-Amylase

The aim of this investigation was to synthesize the adipic acid-modified magnetic nanoparticles for the efficient immobilization of C-terminally lysine-tagged α-amylase (BACΔNC-Lys₇) from thermophilic Bacillus sp. strain TS-23. The carboxylated magnetic nanoparticles were prepared by the simple co-precipitation of Fe³⁺/Fe²⁺ in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy micrographs showed that the carboxylated magnetic nanoparticles remained discrete and had no significant change in size after the binding of BACΔNC-Lys₇. Free enzyme was active in the temperature range of 45-70 °C and had an optimum of 60 °C, whereas the thermal stability of BACΔNC-Lys₇ was improved as a result of immobilization. The immobilized BACΔNC-Lys₇ could be recycled 20 times without a significant loss of the amylase activity and had a better stability during storage with respect to free enzyme. Taken together, the magnetic nanoparticles coated with this functional moiety can be a versatile platform for the effective manipulation of various kinds of engineered proteins.

α-Amylase immobilization onto dye attached magnetic beads: Optimization and characterization

Abstract Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Cibacron Blue F3GA (CB) was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/CB beads (100–140 μm in diameter) carrying 68.3 μmol CB/g polymer were used in α-amylase adsorption studies to assess the effects of pH, initial protein concentration, temperature and ionic strength on enzyme activity. Maximum adsorption capacity of mPHEMA/CB beads was found to be 401 ± 11 mg/g carrier. The adsorbed amounts of α-amylase per unit mass of magnetic beads reached a plateau value at about 1.0 mg/mL at pH 5.0. The optimal pH for free and immobilized α-amylase was 7.0 and 8.0, respectively. The immobilized enzyme exhibited better thermostability than the free one. The free enzyme lost all of its activity within 35 days whereas the immobilized enzyme lost about 27% of its activity during the same period. It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the mPHEMA/CB beads.

SPR based Studies for Pentagalloyl Glucose Binding to α-Amylase

Abstract Astringency is an organoleptic property resulting mostly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers but being typically measured by sensorial panels it turns out subjective and expensive. The main goal of the present work is to develop a sensory system to estimate astringency relying on protein/polyphenol interactions. For this purpose, a model protein was immobilized on a sensory gold surface and its subsequent interaction with polyphenols was measured by Surface Plasma Resonance (SPR). α-amylase and pentagalloyl glucose (PGG) were selected as model protein and polyphenol, respectively. To ensure specific binding between these, various surface chemistries were tested. Carboxylic terminated thiol decreased the binding ability of PGG and allowed covalent attachment of α-amylase to the surface. The pH 5 was the optimal condition for α-amylase immobilization on the surface. Further studies focus on Localized SPR sensor and application to wine samples, providing objectivity when compared to a trained panel.

Immobilization of α-amylase from germinated mung beans (Vigna radiata) on Fuller’s earth by adsorption

A simple, inexpensive and fast method for immobilizing α-amylase from mung bean (Vigna radiata) on Fuller’s earth was developed. For best immobilization (81% activity) the conditions were optimized with activation pH of 5.5 and 350 mg of Fuller’s earth with 6 mg/ml of protein. The optimum pH was slightly shifted towards alkaline side from 5.5 to 5.7, whereas the optimum temperature (65°C) remained unchanged. There was no significant change in Michaelis constant (K m), however, maximum velocity (V max) decreased by four fold upon immobilization. The immobilized enzyme showed an increase in half-life (60 days) and approximately 75% activity remained after five reuses. There was practically no leaching of enzyme from the adsorbent over a period of 20 days. α-Amylase immobilization has potential applications in food, cosmetics, biomedical, and pharmaceuticals industries.

Immobilization of enzymes into self-assembled iron(III) hydrous oxide nano-scaffolds: A bio-inspired one-pot approach to hybrid catalysts

A simple bio-inspired one-pot procedure for the immobilization of α-amylase into maturing hybrid iron(III) hydrous oxide nanostructures is described. The method resorts to the urease mediated decomposition of urea to induce the homogeneous precipitation of amylase-iron(III) hydrous oxide ensembles. Appropriate setting of the synthesis parameters, which control the shape and texture of the resulting hybrid nanostructures, is key to amylase entrapment. Highly efficient hybrid catalysts were prepared at the lowest urease concentration (0.5 mg/mL), where spherical 100 nm size hybrid iron(III) hydrous oxide ensembles formed; their amylase load depended on the enzyme concentration, in a michaelian fashion. Their specific activity is nearly that of free amylase. These catalysts are reusable, with no loss of performance, and substantially more active than the free enzyme at extreme pHs and temperatures. The high efficiency of the hybrid ensembles is ascribed to their open structure, high enzyme loading, and negligible amylase inactivation.

α-Amylase immobilization on the silica nanoparticles for cleaning performance towards starch soils in laundry detergents

In this study, α-amylase was immobilized on silica nanoparticles and immobilized α-amylase was used in formulation of detergent powder for enhancing removal of starch soils. Detergent products contain very components which may affect the free enzyme activity and stability. Also various factors such as temperature, pH and humidity reduced enzyme activity and cleaning efficiency. Therefore the effect of enzyme immobilization on the removal of starch based soil was investigated on cotton fabrics as the model soil. The effect of temperature and humidity on stability of free and immobilized enzyme was compared. It was found that the immobilized enzyme increased cleaning efficiency toward starch soil removal on cotton fabrics, whereas free enzyme imposed a small effect on the enzymatic activity towards the same soil substrates. In addition, stability immobilized enzyme against temperature and humidity was much more than free enzyme.

Immobilization of Soybean α-amylase on Gelatin and its Application as a Detergent Additive

Immobilization of Soybean α-amylase on Gelatin and its Application as a Detergent Additive

Carboxymethyl tamarind gum–silica nanohybrids for effective immobilization of amylase

Abstract Carboxymethyl tamarind gum initiated and catalyzed sol–gel polymerization of tetramethoxysilane produced polysaccharide nanohybrids which were efficient in immobilizing α-amylase for starch hydrolysis. The nanohybrids have been characterized by FTIR, XRD, SEM, TGA and BET analyses. The optimum nanohybrid sample (C) in terms of α-amylase immobilization was synthesized when water (H 2 O), tetramethoxysilane (TMOS) and methanol (MeOH) were used in 17:1:1 ratio at fixed template amount (0.5 g of carboxymethyl tamarind gum). Enzyme immobilization efficiency of “C” was further enhanced on calcination in nitrogen atmosphere, the optimum calcination temperature being 400 °C (C4). Nanohybrid (C4) impregnated with 40 μg of α-amylase was used for hydrolyzing soluble potato starch to glucose syrup at different temperatures, pH values and enzyme concentrations as these parameters affect the kinetics of the studied reaction. The optimum pH and temperature for the hydrolysis reaction were pH 5 and 40 °C, respectively. The kinetic parameters, K m (4.261 mg mL −1 ) and V max (2.55 mol mL −1 min −1 ) for the immobilized amylase were found favorable over the respective values obtained for the free enzyme ( K m = 6.269 mg mL −1 , V max = 1.53 mol mL −1 min −1 ). The enzyme activity remained unchanged up to 90 days inside the nanohybrid matrix. The immobilization at the nanohybrid improved the overall stability, affinity and catalytic property of α-amylase.